Our previous work suggests that the protein "Sunday Driver" (syd) organizes a damage surveillance system in peripheral neurons. In this proposal, we will further test the role of syd in conveying vesicle packages and signaling molecules along axons from the injury site back to the cell body to initiate a regenerative response. Our long-term goal is to identify the mechanisms that activate the intrinsic growth capacity of neurons following axonal injury. This knowledge will be critical to enable the design of new strategies to enhance nerve regeneration following injury. To uncover how syd and its associated membrane compartment may serve as what we call an "injury signaling platform", our first aim is to determine that axonal injury regulates syd interaction with the molecular motors. We will perform immunoprecipitations and sucrose velocity gradients from injured and control mouse sciatic nerves to determine whether syd phosphorylation triggered by nerve injury increases syd interaction with the retrograde motor complex dynein/dynactin, and decreases its interaction with the anterograde motor kinesin. To reveal the role of the c-Jun-N-terminal kinase (JNK) in this process, we will use known JNK inhibitors. This approach will allow us to determine whether JNK activity regulates syd axonal transport in vivo. To further dissect the role of syd phosphorylation by JNK, we will use a primary neuronal cell culture approach to determine whether deleting JNK consensus phosphorylation sites within syd sequence affects syd interaction with the motors and its subcellular distribution. Our second aim will focus on determining the mechanisms by which syd and the motors are recruited to axonal membrane compartments. We will first employ an EM approach to determine whether syd is associated with retrogradely transported endosomes in vivo. We will then perform immunoprecipitation, subcellular fractionation and immunofluorescence experiments to test whether small GTPases of the Rab family play a role in the recruitment of syd and motors to membrane compartments. Finally our third aim will assess the role of syd in nerve regeneration. An RNAi approach in cultured primary dorsal root ganglia (DRG) neurons will allow us to test syd function in axonal regeneration in vitro. Using a syd conditional knockout strategy, we will determine whether syd is critical for the regenerative capacity of peripheral neurons in vivo. To test whether syd injury-induced retrograde transport underlies the ability of PNS neurons to regenerate, we will determine whether syd phosphorylation and interaction with the molecular motors is differentially regulated following injury to peripheral vs. central axons. Altogether, this proposal will address whether the intracellular transport of vesicles that possess syd on their membranes participate in mechanisms that sense axonal injury and organize regenerative responses. PUBLIC HEALTH RELEVANCE: While peripheral neurons have a remarkable ability to repair themselves after injury, most parts of the adult central nervous system, comprised of the brain and the spinal cord, fail to extend new axons when damaged. Understanding the cellular events that confer such properties onto peripheral neurons will improve our ability to restore nerve function following spinal cord injury and stroke, as well as in cases of neurodegenerative diseases in which axonal pathologies interrupt the cell-body/synapse connection. Our work is centered on dissecting the molecular mechanisms regulating how information from the site of injury in axons is conveyed to the cell body to initiate a repair program in peripheral nerves.